Field of the Invention
The present invention relates to compositions and methods for modulating cell density signaling.
Related Art
To function correctly, a cell needs a real-time assessment of its environment. A key component of this running tally is cell density—a parameter known to alter both the rates of cell proliferation and the level of cell differentiation. Loss of cell density awareness has long been associated with the onset of cancer and the ability of malignant cells to overgrow the monolayer when grown in cell culture[1]. Similarly, in normal cells the slowdown in growth as cells become dense is associated with the induction of differentiated function[2]. Despite its importance to normal function, how a cell “sees” its neighbors remains an enigma. The sensing mechanism is known to be influenced by high concentrations of growth factors, for instance high serum levels, but this has not shed light on how a cell actually detects the presence of its neighbors[3,4]. Therefore, to better comprehend tissue morphogenesis and stability requires an understanding of how cells signal their presence to each other.
Tendon morphogenesis is highly dependent on cell density signaling. This is because the tissue is over 90% type I collagen and the two parameters controlling local collagen deposition—the rate of cellular collagen production and the cell number—are cell density regulated[5,6]. Cell density regulation of cell proliferation and apoptosis causes a growth plate to form, allowing the cells to deposit an even distribution of collagen along the longitudinal axis of the fibril—a hallmark of this tissue[7].
In cell culture primary avian tendon (PAT) cells from 16 day embryos devote ˜50% of their total protein production to procollagen[8]. This high level is identical to that seen in ovo where tendon development occurs rapidly (˜11 days) enabling the newly hatched chick the ability to walk. To produce high levels of procollagen from a single copy gene and allow rapid regulation puts restrictions on where this pathway can be controlled. Transcription is an unlikely candidate because induction is slow from a single copy gene. When PAT cells are stimulated to make high levels of collagen, there is a 12 h lag before the cells linearly increase the procollagen mRNA levels over a 2.5 day period[5]. Moreover, to maintain the pool of procollagen mRNA, it has to be stable with a half-life of 24 h[9]. So manipulating procollagen mRNA levels is not feasible when the cells are required to make high levels of procollagen. Instead the cell regulates procollagen at a post-translational step. Translation and secretion rates are both tied to formation of a triple helical molecule and this in turn requires hydroxylation of prolines to stabilize this conformation. This can be manipulated by the addition of ascorbate whose only known role in the process is being a reducing agent and this rapidly (<30 min) induces a 6 fold increase in procollagen secretion[10] and a 2 fold increase in procollagen translation (rates reach 6 fold but require a partial increase in mRNA)[5]. In the fully induced state, inhibiting prolyl hydroxylase by chelating ferrous ion causes a rapid drop in procollagen translation (>50% in 2 h)[5]. The enzyme, prolyl hydroxylase, responsible for this rapid regulatory control requires ascorbate to keep its catalytic ferrous ion in a reduced state. The enzyme has two subunits and the level of the alpha subunit is dependent on cell density[11]. How cell density regulates prolyl hydroxylase has not been defined except that the pool of this enzyme increases 5-6 fold while the level of its mRNA remains unchanged[11]. With 5 cysteines the alpha subunit of prolyl hydroxylase would be sensitive to the redox potential of the cell and this has been postulated as a mechanism for signal transduction[11].
Cell density signaling plays a critical role in the formation of high collagen producing tissues such as tendon, ligament, and bone. Knowing its neighbors allows the cell to control its collagen production and proliferation. The net effect in tendon is the formation of a growth plate where cells at the leading edge at moderate density are growing, cells in the middle at high cell density stop growing and make high levels of collagen, and cells at the trailing edge are apoptosing. In the adult tendon the few remaining cells that were on the periphery of the growth plate and never reached high cell density are now at low cell density in a maintenance state. It is not known whether a damaged tendon can be driven to form a new growth plate by injecting a tendon cell density signal and thereby heal a damaged tendon with speed and strength. Identification of and understanding these signals as potent and specific agents that drive the morphogenesis of tissues is needed.